猪细小病毒SYBR GreenⅠ实时定量PCR检测方法的建立  

Development of SYBR Green Ⅰ real-time PCR assay for detection of porcine parvovirus

作　　者：李明凤[1,2] 魏战勇[1] 王学斌[2] 张红英[1] 王亚宾[1] 崔保安[2] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002 [2]河南省动物性食品安全重点实验室,河南郑州450002

出　　处：《浙江农业学报》2009年第3期220-224,共5页Acta Agriculturae Zhejiangensis

基　　金：河南省杰出人才创新基金项目(0621002100)

摘　　要：利用PCR技术扩增出猪细小病毒VP2保守区基因194 bp片段,并克隆到pGEMT载体上,纯化质粒作为PCR检测的标准模板,以10倍梯度稀释质粒为标准模板,进行SYBR GreenⅠ荧光定量PCR扩增并制作标准曲线,建立了猪细小病毒的荧光定量PCR检测方法。该方法检测灵敏度可达1.0×102拷贝/μL,与猪繁殖与呼吸综合征病毒、猪圆环病毒、猪乙型脑炎病毒、猪伪狂犬病毒、猪瘟病毒和猪流感病毒不发生交叉反应,具有良好的特异性和重复性;对10份疑似病料和阴性病料进行了检测,发现10份均为荧光定量PCR阳性,而常规PCR只能检测出7份阳性。结果表明,建立的实时荧光定量PCR具有特异、敏感、快速、定量、重复性好等优点,可用于临床猪细小病毒(PPV)的检测。A 194 bp region of the porcine parvovims VP2 gene was amplified by PCR and cloned into pGEM T vector, named as pGEM-VP2. Serial dilutions of plasmid pGEM-VP2 were used as standard templates for PCR to quantify the virus genomic copy number. We developed a SYBR Green I real-time PCR to detect porcine parvovirus. Sensitivity analysis showed that the developed SYBR Green I real-time PCR could detect 1.0 × 10^2 template/μL. The specificity assay exhibited that negative control and the other porcine pathogens, such as PRRSV, PCV, JEV, PRV, HCV and SIV could not be detected by this PCR. It was showed that 10 positive results could be observed by the real-time PCR for 10 suspicious positive samples, but 7 positive results by normal PCR. The results suggested that as a result of the good sensitivity and specificity of the assay with a rapid and simple procedure, the real-time PCR method would be useful for the clinical diagnosis of porcine parvovirus infection.

关 键 词：猪细小病毒 实时定量PCR 标准曲线 

分 类 号：S858.28[农业科学—临床兽医学] S852.65[农业科学—兽医学]